Mammalian Tumor Susceptibility Genes and their Uses

ABSTRACT

TSG101 is a tumor susceptibility gene whose homozygous functional knock out in fibroblasts leads to transformation and the ability of these cells to form metastatic tumors in nude mice. The cellular transformation that results from inactivation of TSG101 is reversible by restoration of TSG101 function. Decreased expression of TSG101 is associated with the occurrence of certain human cancers, including breast carcinomas. The TSG101 nucleic acid compositions find use in identifying homologous or related proteins and the DNA sequences encoding such proteins; in producing compositions that modulate the expression or function of the protein; and in studying associated physiological pathways. In addition, modulation of the gene activity in vivo is used for prophylactic and therapeutic purposes, such as treatment of cancer, identification of cell type based on expression, and the like. The DNA is further used as a diagnostic for a genetic predisposition to cancer, and to identify specific cancers having mutations in this gene.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.08/585,758, filed Jan. 12, 1996, which claims priority to U.S.provisional patent application no. 60/006,856, filed Nov. 16, 1995, thedisclosures of which are herein incorporated by reference.

INTRODUCTION

1. Technical Field

The field of the subject invention is mammalian genes associated withsusceptibility to tumors.

2. Background

There has been considerable interest in the development of a method foridentifying mammalian cell genes whose concurrent homozygousinactivation de novo leads to a defined phenotype, where multiplealleles of a gene have been inactivated and where it is easy to confirmthat the inactivation results in a phenotype distinguishable from thewild-type. One use of this method is the identification of genesinvolved in tumor susceptibility.

Tumor susceptibility genes may be oncogenes, which are typicallyupregulated in tumor cells, or tumor suppressor genes, which aredown-regulated or absent in tumor cells. Malignancies may arise when atumor suppressor is lost and/or an oncogene is inappropriatelyactivated. When such mutations occur in somatic cells, they result inthe growth of sporadic tumors. Familial predisposition to cancer mayoccur when there is a mutation, such as loss of an allele encoding atumor suppressor gene, present in the germline DNA of an individual. Inthe best characterized familial cancer syndromes, the primary mutationis a loss of function consistent with viability, but resulting inneoplastic change consequent to the acquisition of a second somaticmutation at the same locus.

Extensive studies of the early-onset breast cancer families have led tothe recent identification of two candidate breast cancer suppressorgenes, BRCA1 and BRCA2. Although frequent mutations of BRCA1 or BRCA2have been demonstrated in familial early onset breast cancer, this typeof cancer represents only about 5-10% of all breast malignancies, andthe possible role(s) of BRCA1 and BRCA2 in the remaining 90-95% ofsporadic breast cancers has not been determined.

Deletion and loss of heterozygosity (LOH) of markers in human chromosomeband 11p15 have been shown in a variety of human cancers, including lungcancer, testicular cancer and male germ cell tumor, stomach cancer,Wilms' tumor, ovarian cancer, bladder cancer, myeloid leukemia,malignant astrocytomas and other primitive neuroectodermal tumors, andinfantile tumors of adrenal and liver. About 30% of sporadic breastcarcinomas show a LOH in this region. Since LOH is believed to indicateinactivation of a tumor suppressor gene at the location where LOHoccurs, the frequent LOH found at 11p15 in a variety of human cancerssuggests the presence of either a cluster of tumor suppressor genes or asingle pleiotropic gene in this region.

The clinical importance of these cancers makes the identification ofthis putative tumor suppressor gene of great interest for diagnosis,therapy, and drug screening.

Relevant Literature

Lemke et al. (1993) Glia 7:263-271 describes loss of function mutationsengineered through the expression of antisense RNA from previouslycloned genes and through the insertional inactivation of the P_(o) gene,by homologous recombination in embryonic stem cells; and the generationof P_(o)-deficient mice. Kamano et al. (1990) Leukemia Res. 10:831-839;van der Krol et al. (1988) Biotechniques 6:958; Katsuki et al. (1988)Science 241:593-595; Owens et al. (1991) Development 112:639-649;. andOwens et al. (1991) Neuron 7:565-575 describe changes in cell phenotypeassociated with the expression of antisense RNAs in different celltypes. Giese et al. (1992) Cell, 71:565-576 describes the inactivationof both copies of a gene in a transgenic mouse.

Studies of LOH in Wilms' tumors identified a tumor suppressor locus at11p15, for example see Dowdy et al. (1991) Science 254:293-295. Twofamilial breast cancer genes have been previously described, BRCA1 inMiki et al. (1994) Science 266:66-71, and BRCA2 in Wooster et al. (1995)Nature 378:789-792.

The interaction of stathmin with a coiled coil domain is described inSobel (1991) Trends Biochem. Sci. 16:301-305.

SUMMARY OF THE INVENTION

Mammalian tumor susceptibility genes and methods for theiridentification are provided, including the complete nucleotide sequencesof human TSG101 and mouse tsg101 cDNA. Deletions in TSG101 areassociated with the occurrence of human cancers, for example breastcarcinomas. The cancers may be familial, having as a component of riskan inherited genetic predisposition, or may be sporadic. The TSG101nucleic acid compositions find use in identifying homologous or relatedproteins and the DNA sequences encoding such proteins; in producingcompositions that modulate the expression or function of the protein;and in studying associated physiological pathways. In addition,modulation of the gene activity in vivo is used for prophylactic andtherapeutic purposes, such as treatment of cancer, identification ofcell type based on expression, and the like. The DNA is further used asa diagnostic for a genetic predisposition to cancer, and to identifyspecific cancers having mutations in this gene.

BRIEF DESCRIPTION. OF THE DRAWINGS

FIG. 1 is a diagram of the vectors: (a) pLLGSV; (b) pLLTX; and (c)pRSV-cre.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Mammalian tsg101 gene compositions and methods for their isolation areprovided. Of particular interest are the human and mouse homologs.Certain human cancers show deletions at the TSG101 locus. Many suchcancers are sporadic, where the tumor cells have a somatic mutation inTSG101. The TSG101 genes and fragments thereof, encoded protein, andanti-TSG101 antibodies are useful in the identification of individualspredisposed to development of such cancers, and in characterizing thephenotype of sporadic tumors that are associated with this gene. Tumorsmay be typed or staged as to the TSG101 status, e.g. by detection ofmutated sequences, antibody detection of abnormal protein products, andfunctional assays for altered TSG101 activity. The encoded. TSG101protein is useful in drug screening for compositions that mimic TSG101activity or expression, particularly with respect to TSG101 function asa tumor suppressor in oncogenesis. TSG101 can be used to investigate theinteractions with stathmin and the role the complex plays in theregulation of the cell.

The human TSG101 and mouse tsg101 gene sequences and isolated nucleicacid compositions are provided. In identifying the human and mouseTSG101/tsg101 genes, the novel gene discovery approach “randomhomozygous knock out” was utilized. A retroviral gene search vectorcarrying a reporter gene was used to select and identify cellscontaining the vector integrated into target transcriptionally activechromosomal DNA regions, behind chromosomal promoters. 5′ to and inreverse orientation to the reporter gene was a regulated promoter withno transcription activity, but which could be highly activated by atransactivator. The system generates large amounts of antisense RNA,which interacts with both alleles of the target gene. Cells transfectedwith the search vector were further transfected with a plasmid encodinga transactivator. The cells were plated to select for genes whoseinactivation led to cellular transformation. While control cellpopulations formed no colonies in soft agar, the transactivated cellsproduced 20 colonies. One of these clones was shown to be highlytumorigenic in nude mice. mRNA selection, using a primer specific forthe reporter gene, was used to isolate mRNA from the target gene. ThemRNA was then used to generate a cDNA clone, which was further used inhybridization screening to isolate the full-length mouse tsg101 cDNA.

To obtain the human homolog of mouse tsg101, the mouse cDNA sequence wasused to query dbEST. Ten human partial cDNA sequences included in thedatabase showed 85% to 95% identity to mouse tsg101. A conservedsequence was used to design primers that amplify segments of humanTSG101 cDNA, employing total DNA isolated from a human cDNA library astemplate. The TSG101 gene has been mapped to human chromosome sub-bands11p15.1-15.2, and is closely linked to the Sequence Tagged Site (STS)markers D11S921 through D11S1308 (a detailed map of human genome markersmay be found in Dib et el. (1996) Nature 280:152;http://www.genethon.fr).

The full length human cDNA contains an 1140 bp open reading frame,encoding a 380 amino acid protein. The human and mouse cDNAs are 86%identical at the nucleotide level. The predicted proteins are 94%identical and are distinguished by 20 amino acid mismatches and one gap.A coiled-coil domain (human TSG101 aa 231-302) and a proline-rich domain(human TSG101 aa 130-205, 32% proline) typical of the activation domainsof transcription factors are highly conserved between the human andmouse proteins, with only one amino acid mismatch in each of the twodomains. The leucine zipper motif in the coiled-coil domain of the humanTSG101 protein is identical to the one in the mouse protein.

DNA from a tumor that is suspected of being associated with TSG101 isanalyzed for the presence of an oncogenic mutation in the TSG101 gene.Sporadic tumors associated with loss of TSG101 function include a numberof carcinomas known to have deletions in the region of human chromosome11p15, e.g. carcinomas Of the breast, lung cancer, testicular cancer andmale germ cell tumor, stomach cancer, Wilms' tumor, ovarian cancer,bladder cancer, myeloid leukemia, malignant astrocytomas and otherprimitive neuroectodermal tumors, and infantile tumors of adrenal andliver.

Characterization of sporadic tumors will generally require analysis oftumor cell DNA, conveniently with a biopsy sample. Where metastasis hasoccurred, tumor cells may be detected in the blood. Of particularinterest is the detection of deletions in the TSG101 gene, e.g. byamplification of the region and size fractionation of the amplificationproduct; restriction mapping, etc. Screening of tumors may also be basedon the functional or antigenic characteristics of the protein.Immunoassays designed to detect the normal or abnormal TSG101 proteinmay be used in screening. Alternatively, functional assays, e.g. assaysbased on detecting changes in the stathmin pathway mediated by TSG101,may be performed.

A wide range of mutations are found, up to and including deletion of theentire short arm of chromosome 11. Specific mutations of interestinclude any mutation that leads to oncogenesis, including insertions,substitutions and deletions in the coding region sequence, introns thataffect splicing, promoter or enhancer that affect the activity andexpression of the protein. A “normal” sequence of TSG101 is provided inSEQ ID NO:3 (human). In many cases, mutations disrupt the coiled coildomain, resulting in a protein that is truncated or has a deletion inthis region. Other mutations of interest may affect the proline richdomain, or other conserved regions of the protein. The leucine zipperwithin the coiled coil domain is also of particular interest.Biochemical studies may be performed to confirm whether a candidatesequence variation in the TSG101 coding region or control regions isoncogenic. For example, oncogenicity activity of the mutated TSG101protein may be determined by its ability to complement a loss of TSG101activity in 3T3 cells, by binding studies with stathmin, etc.

The TSG101 gene may also be used for screening of patients suspected ofhaving a genetic predisposition to TSG101-associated tumors, where thepresence of a mutated TSG101 sequence confers an increasedsusceptibility to cancer. Diagnosis is performed by protein, DNAsequence, PCR screening, or hybridization analysis of any convenientsample from a patient, e.g. biopsy material, blood sample, scrapingsfrom cheek, etc. A typical patient genotype will have an oncogenicmutation on one chromosome. When the normal copy of TSG101 is lost,leaving only the reduced function mutant copy, abnormal cell growth isthe result.

Prenatal diagnosis may be performed, particularly where there is afamily history of the disease, e.g. an affected parent or sibling. Asample of fetal DNA, such as an amniocentesis sample, fetal nucleated orwhite blood cells isolated from maternal blood, chorionic villus sample,etc. is analyzed for the presence of the predisposing mutation.Alternatively, a protein based assay, e.g. functional assay orimmunoassay, is performed on fetal cells known to express TSG101.

The DNA sequence encoding TSG101 may be cDNA or genornic DNA or afragment thereof. The term “TSG101 gene” shall be intended to mean theopen reading frame encoding specific TSG101 polypeptides, as well asadjacent 5′ and 3′ non-coding nucleotide sequences involved in theregulation of expression, up to about 1 kb beyond the, coding region, ineither direction. The gene may be introduced into an appropriate vectorfor extrachromosomal maintenance or for integration into the host.

The term “cDNA” as used herein is intended to include all nucleic acidsthat share the arrangement of sequence elements found in native maturemRNA species, where sequence elements are exons, 3′ and 5′ non-codingregions. Normally mRNA species have contiguous exons, with theintervening introns deleted, to create a continuous open reading frameencoding TSG101.

The genomic TSG101 sequence has non-contiguous open reading frames,where introns interrupt the coding regions. A genomic sequence ofinterest comprises the nucleic acid present between the initiation codonand the stop codon, as defined in the listed sequences, including all ofthe introns that are normally present in a native chromosome. It mayfurther include the 3′ and 5′ untranslated regions found in the maturemRNA. It may further include specific transcriptional and translationalregulatory sequences, such as promoters, enhancers, etc., includingabout 1 kb of flanking genomic DNA at either the 5′ or 3′ end of thecoding region. The genomic DNA may be isolated as a fragment of 50 kbpor smaller; and substantially free of flanking chromosomal sequence.

The nucleic acid compositions of the subject invention encode all or apart of the subject polypeptides. Fragments may be obtained of the DNAsequence by chemically synthesizing oligonucleotides in accordance withconventional methods, by restriction enzyme digestion, by PCRamplification, etc. For the most part, DNA fragments will be of at least15 nt, usually at least 18 nt, more usually at least about 50 nt. Suchsmall DNA fragments are useful for hybridization screening, etc. LargerDNA fragments, i.e. greater than 100 bp, usually greater than 500 bp,are useful for production of the encoded polypeptide. Single strandedoligonucleotides of from about 18 to 35 nt in length are useful for PCRamplifications. For use in amplification reactions, such as PCR, a pairof primers will be used. The exact composition of the primer sequencesis not critical to the invention, but for most applications the primerswill hybridize to the subject sequence under stringent conditions, asknown in the art. It is preferable to chose a pair of primers that willgenerate an amplification product of at least about 50 nt, preferably atleast about 100 nt. Algorithms for the selection of primer sequences aregenerally known, and are available in commercial software packages.Amplification primers hybridize to complementary strands of DNA, andwill prime towards each other.

The TSG101 genes are isolated and obtained in substantial purity,generally as other than an intact mammalian chromosome. Usually, the DNAwill be obtained substantially free of other nucleic acid sequences thatdo not include a TSG101 sequence or fragment thereof, generally being atleast about 50%, usually at least about 90% pure and are typically“recombinant”, i.e. flanked by one or more nucleotides with which it isnot normally associated on a naturally occurring chromosome.

The DNA sequences are used in a variety of ways. They may be used asprobes for identifying other tsg101 genes. Mammalian homologs havesubstantial sequence similarity to the subject sequences, i.e. at least75%, usually at least 90%, more usually at least 95% sequence identitywith the nucleotide sequence of the subject DNA sequence. Sequencesimilarity is calculated based on a reference sequence, which may be asubset of a larger sequence, such as a conserved motif, coding region,flanking region, etc. A reference sequence will usually be at leastabout 18 nt long, more usually at least about 30 nt long, and may extendto the complete sequence that is being compared. Algorithims forsequence analysis are known in the art, such as BLAST, described inAltschul et al. (1990) J Mol Biol 215:403-10.

Nucleic acids having sequence similarity are detected by hybridizationunder low stringency conditions, for example, at 50° C. and 10×SSC (0.9M saline/0.09 M sodium citrate) and remain bound when subjected towashing at 55° C. in 1×SSC. By using probes, particularly labeled probesof DNA sequences, one can isolate homologous or related genes. Thesource of homologous genes may be any mammalian species, e.g. primatespecies; murines, such as rats and mice; canines; felines; bovines;ovines; equines; etc.

The DNA may also be used to identify expression of the gene in abiological specimen. The manner in which one probes cells for thepresence of particular nucleotide sequences, as genomic DNA or RNA, iswell-established in the literature and does not require elaborationhere. Conveniently, a biological specimen is used as a source of mRNA.The mRNA may be amplified by RT-PCR, using reverse transcriptase to forma complementary DNA strand, followed by polymerase chain reactionamplification using primers specific for the subject DNA sequences.Alternatively, the mRNA sample is separated by gel electrophoresis,transferred to a suitable support, e.g. nitrocellulose, and then probedwith a fragment of the subject DNA as a probe. Other techniques may alsofind use. Detection of mRNA having the subject sequence is indicative ofTSG101 gene expression in the sample.

The subject nucleic acid sequences may be modified for a number ofpurposes, particularly where they will be used intracellularly, forexample, by being joined to a nucleic acid cleaving agent, e.g. achelated metal ion, such as iron or chromium for cleavage of the gene;as an antisense sequence; or the like. Modifications may includereplacing oxygen of the phosphate esters with sulfur or nitrogen,replacing the phosphate with phosphoramide, etc.

A number of methods are available for analyzing genomic DNA sequencesfor the presence of mutations. Where large amounts of DNA are available,the genomic DNA is used directly. Alternatively, the region of interestis cloned into a suitable vector and grown in sufficient quantity foranalysis, or amplified by conventional techniques, such as thepolymerase chain reaction (PCR). The use of the polymerase chainreaction is described in Saiki et al. (1985) Science 239:487, and areview of current techniques may be found in Sambrook, et al. MolecularCloning: A Laboratory Manual, CSH Press 1989, pp.14.2-14.33.

PCR is particularly useful for detection of oncogenic mutations. In manycases such mutations involve a deletion at the TSG101 locus. Forexample, primers specific for TSG101 are used to amplify all or part ofthe gene. The amplification products are then analyzed for size, where adeletion will result in a smaller than expected product. Where thedeletion is very large, there may be a complete absence of the specificamplification product. Alternatively, analysis may be performed on mRNAfrom a cell sample, where the RNA is converted to cDNA, and thenamplified (RT-PCR).

A detectable label may be included in the amplification reaction.Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate(FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin,6-carboxyfluorescein (6-FAM),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE),6-carboxy-X-rhodamine (ROX),6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), radioactivelabels, e.g. ³²P, ³⁵S, ³H; etc. The label may be a two stage system,where the amplified DNA is conjugated to biotin, haptens, etc.

having a high affinity binding partner, e.g. avidin, specificantibodies, etc., where the binding partner is conjugated to adetectable label. The label may be conjugated to one or both of theprimers. Alternatively, the pool of nucleotides used in theamplification is labeled, so as to incorporate the label into theamplification product.

The amplified or cloned fragment may be sequenced by dideoxy or othermethods, and the sequence of bases compared to the normal TSG101sequence. Hybridization with the variant, oncogenic sequence may also beused to determine its presence, by Southern blots, dot blots, etc.Single strand conformational polymorphism (SSCP) analysis, denaturinggradient gel electrophoresis (DGGE), and heteroduplex analysis in gelmatrices are used to detect conformational changes created by DNAsequence variation as alterations in electrophoretic mobility. Thehybridization pattern of a control and variant sequence to an array ofoligonucleotide probes immobilised on a solid support, as described inWO 95/11995, may also be used as a means of detecting the presence ofvariant sequences. Alternatively, where an oncogenic mutation creates ordestroys a recognition site for a restriction endonuclease, the fragmentis digested with that endonuclease, and the products size fractionatedto determine whether the fragment was digested. Fractionation isperformed by gel electrophoresis, particularly acrylamide or agarosegels.

The subject nucleic acids can be used to generate transgenic animals orsite specific gene modifications in cell lines. The modified cells oranimals are useful in the study of TSG101 function and regulation. Forexample, a series of small deletions and/or substitutions may be made inthe TSG101 gene to determine the role of different exons in oncogenesis,signal transduction, etc. One may also provide for expression of theTSG101. gene or variants thereof in cells or tissues where it is notnormally expressed or at abnormal times of development. In addition, byproviding expression of TSG101 protein in cells in which it is otherwisenot normally produced, one can induce changes in cell behavior.

DNA constructs for homologous recombination will comprise at least aportion of the TSG101 gene with the desired genetic modification, andwill include regions of homology to the target locus. Alternatively,constructs may that do not target to the native locus, but integrate atrandom sites in the genome. Conveniently, markers for positive andnegative selection are included. Methods for generating cells havingtargeted gene modifications through recombination are known in the art.For various techniques for transfecting mammalian cells, see Keown etal. (1990) Methods in Enzymology 185:527-537.

For embryonic stem (ES) cells, an ES cell line may be employed, or EScells may be obtained freshly from a host, e.g. mouse, rat, guinea pig,etc. Such cells are grown on an appropriate fibroblast-feeder layer orgrown in the presence of leukemia inhibiting factor (LIF). When ES cellshave been transformed, they may be used to produce transgenic animals.After transformation, the cells are plated onto a feeder layer in anappropriate medium. Cells containing the construct may be detected byemploying a selective medium. After sufficient time for colonies togrow, they are picked and analyzed for the occurrence of homologousrecombination. Those colonies that show homologous recombination maythen be used for embryo manipulation and blastocyst injection.Blastocysts are obtained from 4 to 6 week old superovulated females. TheES cells are trypsinized, and the modified cells are injected into theblastocoel of the blastocyst. After injection, the blastocysts arereturned to each uterine horn of pseudopregnant females. Females arethen allowed to go to term and the resulting litters screened for mutantcells having the construct. By providing for a different phenotype ofthe blastocyst and the ES cells, chimeric progeny can be readilydetected.

The chimeric animals are screened for the presence of the modified geneand males and females having the modification are mated to producehomozygous progeny. If the gene alterations cause lethality at somepoint in development, tissues or organs can be maintained as allogeneicor congenic graft or transplants, or in in vitro culture. The transgenicanimals may be any non-human mammal, such as laboratory animals,domestic animals, etc. The transgenic animals may be used in functionalstudies, drug screening, etc., e.g. to determine the effect of acandidate drug on tumor cells.

The subject gene may be employed for producing all or portions of theTSG101 protein. Peptides of interest include the coiled-coil domain (aa231-302) and the proline-rich domain (aa 130-205). For expression, anexpression cassette may be employed, providing for a transcriptional andtranslational initiation region, which may be inducible or constitutive,the coding region under the transcriptional control of thetranscriptional initiation region, and a transcriptional andtranslational termination region. Various transcriptional initiationregions may be employed which are functional in the expression host.

The peptide may be expressed in prokaryotes or eukaryotes in accordancewith conventional ways, depending upon the purpose for expression. Forlarge scale production of the protein, a unicellular organism or cellsof a higher organism, e.g. eukaryotes such as vertebrates, particularlymammals, may be used as the expression host, such as. E. coli, B,subtilis, S. cerevisiae, and the like. In many situations, it may bedesirable to express the TSG101 gene in a mammalian host, whereby theTSG101 protein will be glycosylated.

With the availability of the protein in large amounts by employing anexpression host, the protein may be isolated and purified in accordancewith conventional ways. A lysate may be prepared of the expression hostand the lysate purified using HPLC, exclusion chromatography, gelelectrophoresis, affinity chromatography, or other purificationtechnique; The purified protein will generally be at least about 80%pure, preferably at least about 90% pure, and may be up to and including100% pure. By pure is intended free of other proteins, as well ascellular debris.

TSG101 polypeptides are useful in the investigation of the stathminsignaling pathway, which is involved in the regulation and relay ofdiverse signals associated with cell growth and differentiation. Thecoiled coil domain of TSG101 interacts with stathmin. The structure ofTSG101 indicates that it is a transcription factor, which may act as adownstream effector of stathmin signaling. The normal and mutated formsof TSG101 polypeptides may be used for binding assays with otherproteins, to detect changes in phosphorylation, etc. that may affectthis pathway. Yeast has been shown to be a powerful tool for studyingprotein-protein interactions through the two hybrid system described inChien et al. (1991) P.N.A.S. 88:9578-9582.

Binding assays of TSG101 to DNA may be performed in accordance withconventional techniques for DNA footprinting, to determine the sequencemotifs that are recognized by TSG101. In vitro transcription assays maybe used, to determine how complexes comprising polymerase andtranscriptional activation factors are affected by the presence ofTSG101.

The polypeptide is used for the production of antibodies, where shortfragments provide for antibodies specific for the particularpolypeptide, whereas larger fragments or the entire gene allow for theproduction of antibodies over the surface of the polypeptide or protein.Antibodies may be raised to the normal or mutated forms of TSG101. Thecoiled coil, leucine zipper and proline rich domains of the protein areof interest as epitopes, particularly to raise antibodies that recognizecommon changes found in oncogenic TSG101. Antibodies may be raised toisolated peptides corresponding to these domains, or to the nativeprotein. Antibodies that recognize TSG101 are useful in diagnosis,typing and staging of human tumors, e.g. breast carcinomas.

Antibodies are prepared in accordance with conventional ways, where theexpressed polypeptide or protein may be used as an immunogen, by itselfor conjugated to known immunogenic carriers, e.g. KLH, pre-S HBsAg,other viral or eukaryotic proteins, or the like. Various adjuvants maybe employed, with a series of injections, as appropriate. For monoclonalantibodies, after one or more booster injections, the spleen may beisolated, the splenocytes immortalized, and then screened for highaffinity antibody binding. The immortalized cells, e.g. hybridomas,producing the desired antibodies may then be expanded. For furtherdescription, see Monoclonal Antibodies: A Laboratory Manual, Harlow andLane eds., Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y.,1988. If desired, the mRNA encoding the heavy and light chains may beisolated and mutigenized by cloning in E. coli, and the heavy and lightchains may be mixed to further enhance the affinity of the antibody.

The antibodies find particular use in diagnostic assays for carcinomasand other tumors associated with mutations in TSG101. Staging, detectionand typing of tumors may utilize a quantitative immunoassay for thepresence or absence of normal TSG101. Alternatively, the presence ofmutated forms of TSG101 may be determined. A reduction in normal TSG101and/or presence of abnormal TSG101 is indicative that the tumor isTSG101-associated.

A sample is taken from a patient suspected of having a TSG101-associatedtumor. Samples, as used herein, include biological fluids such as blood,cerebrospinal fluid, tears, saliva, lymph, dialysis fluid and the like,organ or tissue culture derived fluids; and fluids extracted fromphysiological tissues. Also included in the term are derivatives andfractions of such fluids. Biopsy samples are of particular interest,e.g. carcinoma samples, organ tissue fragments, etc. Where metastasis issuspected, blood samples may be preferred. The number of cells in asample will generally be at least about 10³, usually at least 10⁴ moreusually at least about 10⁵. Usually a lysate of the cells is prepared.

Diagnosis may be performed by a number of methods. The different methodsall determine the absence or presence of normal or abnormal TSG101 inpatient cells suspected of having a mutation in TSG101. For example,detection may utilize staining of histological sections, performed inaccordance with conventional methods. The antibodies of interest areadded to the cell sample, and incubated for a period of time sufficientto allow binding to the epitope, usually at least about 10 minutes. Theantibody may be labeled with radioisotopes, enzymes, fluorescers,chemiluminescers, or other labels for direct detection. Alternatively, asecond stage antibody or reagent is used to amplify the signal. Suchreagents are well-known in the art. For example, the primary antibodymay be conjugated to biotin, with horseradish peroxidase-conjugatedavidin added as a second stage reagent. Final detection uses a substratethat undergoes a color change in the presence of the peroxidase. Theabsence or presence of antibody binding may be determined by variousmethods, including microscopy, spectrophometry, scintillation counting,etc.

An alternative method for diagnosis depends on the in vitro detection ofbinding between antibodies and TSG101 in a lysate. Measuring, theconcentration of TSG101 binding in a sample or fraction thereof may beaccomplished by a variety of specific assays. A conventional sandwichtype assay may be used. For example, a sandwich assay may first attachTS G101-specific antibodies to an insoluble surface or support. Theparticular manner of binding is not crucial so long as it is compatiblewith the reagents and overall methods of the invention. They may bebound to the plates covalently or non-covalently, preferablynon-covalently.

The insoluble supports may be any compositions to which polypeptides canbe bound, which is readily separated from soluble material, and which isotherwise compatible with the overall method. The surface of suchsupports may be solid or porous and of any convenient shape. Examples ofsuitable insoluble supports to which the receptor is bound includebeads, e.g. magnetic beads, membranes and microtiter plates. These aretypically made of glass, plastic (e.g. polystyrene), polysaccharides,nylon or nitrocellulose. Microtiter plates are especially convenientbecause a large number of assays can be carried out simultaneously,using small amounts of reagents and samples.

Patient sample lysates are then added to separately assayable supports(for example, separate wells of a microtiter plate) containingantibodies. Preferably, a series of standards, containing knownconcentrations of normal and/or abnormal TSG101 is assayed in parallelwith the samples or aliquots thereof to serve as controls. Preferably,each sample and standard will be added to multiple wells so that meanvalues can be obtained for each. The incubation time should besufficient for binding, generally, from about 0.1 to 3 hr is sufficient.After incubation, the insoluble support is generally washed of non-boundcomponents. Generally, a dilute non-ionic detergent medium at anappropriate pH, generally 7-8, is used as a wash medium. From one to sixwashes may be employed, with sufficient volume to thoroughly washnon-specifically bound proteins present in the sample.

After washing, a solution containing a second antibody is applied. Theantibody will bind TSG101 with sufficient specificity such that it canbe distinguished from other components present. The second antibodiesmay be labeled to facilitate direct, or indirect quantification ofbinding. Examples of labels that permit direct measurement of secondreceptor binding include radiolabels, such as ³H or ¹²⁵I, fluorescers,dyes, beads, chemilumninescers, colloidal particles, and the like.Examples of labels which permit indirect measurement of binding includeenzymes where the substrate may provide for a colored or fluorescentproduct. In a preferred embodiment, the antibodies are labeled with acovalently bound enzyme capable of providing a detectable product signalafter addition of suitable substrate. Examples of suitable enzymes foruse in conjugates include horseradish peroxidase, alkaline phosphatase,malate dehydrogenase and the like. Where not commercially available,such antibody-enzyme conjugates are readily produced by techniques knownto those skilled in the art. The incubation time should be sufficientfor the labeled ligand to bind available molecules. Generally, fromabout 0.1 to 3 hr is sufficient, usually 1 hr sufficing.

After the second binding step, the insoluble support is again washedfree of non-specifically bound material. The signal produced by thebound conjugate is detected by conventional means. Where an enzymeconjugate is used, an appropriate enzyme substrate is provided so adetectable product is formed.

Other immunoassays are known in the art and may find use as diagnostics.Ouchterlony plates provide a simple determination of antibody binding.Western blots may be performed on protein gels or protein spots onfilters, using a detection system specific for TSG101 as desired,conveniently using a labeling method as described for the sandwichassay.

By providing for the production of large amounts of TSG101 protein, onecan identify ligands or substrates that bind to, modulate or mimic theaction of TSG101. Areas of investigation include the development ofcancer treatments. Drug screening identifies agents that provide areplacement for TSG101 function in abnormal cells. The role of TSG101 asa tumor suppressor indicates that agents which mimic its function willinhibit the process of oncogenesis. Of particular interest are screeningassays for agents that have a low toxicity for human cells. A widevariety of assays may be used for this purpose, including labeled invitro protein-protein binding assays, electrophoretic mobility shiftassays, immunoassays for protein binding, and the like. The purifiedprotein may also be used for determination of three-dimensional crystalstructure, which can be used for modeling intermolecular interactions,transcriptional regulation function, etc.

The term “agent” as used herein describes any molecule, protein, orpharmaceutical with the capability of altering or mimicking thephysiological function of TSG101. Generally a plurality of assaymixtures are run in parallel with different agent concentrations toobtain a differential response to the various concentrations. Typically,one of these concentrations serves as a negative control, i.e. at zeroconcentration or below the level of detection.

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 50 and less than about 2,500 daltons.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

Where the screening assay is a binding assay, one or more of themolecules may be joined to a label, where the label can directly orindirectly provide a detectable signal. Various labels includeradioisotopes, fluorescers, chemiluminescers, enzymes, specific bindingmolecules, particles, e.g. magnetic particles, and the like. Specificbinding molecules include pairs, such as biotin and streptavidin,digoxin and antidigoxin etc. For the specific binding members, thecomplementary member would normally be labeled with a molecule thatprovides for detection, in accordance with known procedures.

A variety of other reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc that are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Reagentsthat improve the efficiency of the assay, such as protease inhibitors,nuclease inhibitors, anti-microbial agents, etc. may be used. Themixture of components are added in any order that provides for therequisite binding. Incubations are performed at any suitabletemperature, typically between 4 and 40° C. Incubation periods areselected for optimum activity, but may also be optimized to facilitaterapid high-throughput screening. Typically between 0.1 and 1 hours willbe sufficient.

Other assays of interest detect agents that mimic TSG101 function. Forexample, candidate agents are added to a cell that lacks functionalTSG101, and screened for the ability to reproduce TSG101 function, e.g.prevent growth of 3T3 cells in soft agar.

The compounds having the desired pharmacological activity may beadministered in a physiologically acceptable carrier to a host fortreatment of cancer attributable to a defect in tsg101 function. Theinhibitory agents may be administered in a variety of ways, orally,topically, parenterally e.g. subcutaneously, intraperitoneally,intravascularly, etc. Topical treatments are of particular interest.Depending upon the manner of introduction, the compounds may beformulated in a variety of ways. The concentration of therapeuticallyactive compound in the formulation may vary from about 0.1-100 wt. %.

The pharmaceutical compositions can be prepared in various forms, suchas granules, tablets, pills, suppositories, capsules, suspensions,salves, lotions and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically-active compounds.Diluents known to the art include aqueous media, vegetable and animaloils and fats. Stabilizing agents, wetting and emulsifying agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.

The gene may also be used for gene therapy. Vectors useful forintroduction of the gene include plasmids and viral vectors. Ofparticular interest are retroviral-based vectors, e.g. moloney murineleukemia virus and modified human immunodeficiency virus; adenovirusvectors, etc. Gene therapy may be used to treat cancerous lesions, anaffected fetus, etc., by transfection of the normal gene into suitablecells. A wide variety of viral vectors can be employed for transfectionand stable integration of the gene into the genome of the cells.Alternatively, micro-injection may be employed, fusion, or the like forintroduction of genes into a host cell. See, for example, Dhawan et al.(1991) Science 254:1509-1512 and Smith et al. (1990) Molecular andCellular Biology 3268-3271.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1

The method described below allows for the identification and isolationof new genes involved in the regulation of cell growth anddifferentiation. Preparation of constructs, methods for mammalian celltransformation, assays for uncontrolled cell growth, and methods foridentifying the new gene are provided.

Results

Experimental Approach and Construction of Gene Search Vectors. Theexperimental strategy used is shown schematically in FIG. 1. pLLGSV, aretroviral gene search vector derived from self-inactivating Moloneymurine leukemia virus (MLV) (Hawley et al., PNAS USA (1987)84:2406-2410; Brenner at al., PNAS USA (1989) 86:5517-5521) carries theβ-geo (Friedrich and Soriano, Genes & Develop. (1991) 5:1513-1523)reporter gene. This reporter, a fusion of the E. coil lacZ andaminoglycoside phosphotransferase (aph or “neo”) genes, encodesresistance to the antibiotic G418, which was used to select and identifycells containing virus integrated into transcriptionally activechromosomal DNA regions behind chromosomal promoters. Anadenovirus-derived splice acceptor (Friedrich and Soriano, 1991 supra)was inserted at the 5′ end of β-geo to enhance the fusion of β-geo mRNAto upstream transcripts encoded by chromosomally-encoded exons. 5′ to,and in reverse orientation to β-geo, is a regulated promoter formed byfusion of the SV40 early T antigen minimal promoter sequence to 14E.coli lacZ operators (Labow et al., Mol. Cell. Biol. (1990)10:3343-3356); this promoter has no transcription activity, but can behighly activated in trans by a transactivator, Lap348 (Lebow et al.,1990, supra), containing the operator-binding domain of the E coli /adrepressor and the herpes simplex virus transactivation domain VP16. Thesystem was designed to generate large amounts of antisense RNA, whichinteract not only with the sense RNA encoded by the allele with theintegrated gene search vector, but also with the sense RNA encoded byother allele(s) of the same gene.

pLLGAV was first transfected into helper cells (GP+E-86) to generateinfectious viruses to infect NIH3T3 cells. A population of G418resistant NIH3T3 cells, containing the pLLGSV vector integrated attranscriptionally active sites behind chromosomal promoters throughoutthe 3T3 cell genome, were transfected with transactivator vector pLLTX.pLLTX encodes both the Lap348 and HyTK, a fusion of a hygromycinresistance (hyg) gene and the herpes simplex virus thymidine kinase (TK)gene (Lupton at al., Mol. Cell. Biol. (1991) 11:3374-3378).Transfectants expressing HyTK are resistant to hyg but sensitive togancyclovir (gcv), which specifically kills cells expressing herpes TK.In contrast, in the absence of HyTK expression, calls are hyg-sensitiveand gcv-resistant. Two lox sites from bacteriophage P1 flanking thetransactivator and HyTK genes allow excision of the Lap348/HyTK segmentfrom -chromosomes of cells by Cre, a lox-specific recombinase (Sauer andHenderson, Nature (1989) 298:447-451) expressed from pRSV-cre introducedinto hyg resistant cells by electoporation. Cells in which theLap348/HyTK segment has been excised, and in which the regulatedpromoter consequently has been turned off, are detected by theirresistance to gcv.

hyg resistant NIH3T3 cells were plated in 0.5% agarose to select fortransformation phenotype, i.e., to select genes whose inactivation maycontribute to cellular transformation. Excision of LAP348 fromtransformed cells by Cre generated transactivator deleted clones.Comparing the phenotypes of the cells with transactivator present andcells with transactivator deleted, further confirms that cellulartransformation results from transactivator generated antisense RNA.Cells with transactivator deleted can be used for cloning of the genecontaining the gene search vector.

Isolation of Clones Showing Transformed Phenotype. 2.5×10⁸ NIH 3T3 cellswere infected with viral supernatant from a culture of apLLGSV-transfected helper cell clone selected for its ability to producea high titer of infectious virus. Infected cells containingchromosomally integrated pLLGSV were either selected on plates for G418resistance or collected by fluorescence-activated cell sorting (Brenneret al., 1989, supra) for β-galactosidase activity; the cell populationobtained by either method showed variable degrees of deep blue stainingby X-gal. A pool of more than 5×10⁶ clones containing retroviralintegrations selected for G418 resistance was transfected with thetransactivator vector pLLTX by electroporation; colonies selected forhyg resistance were pooled and plated in 0.5% agarose. Whereas no cellsin a similarly-sized uninfected NIH 3T3 population formed colonies onthis concentration of agarose, the pLLGSV infected population produced20 colonies. One of these clones, SL6 was expanded into cell line, whichwas transfected with pRSV-cre to generate cells with deletedtransactivator (SL6ΔT cells. Both SL6 and SL6ΔT cells were injected intonude mice subcutaneously, where only SL6 cells were highly tumorigenic.Although SL6ΔT cells produced a small tumor in one mouse, neithercontrol NIH3T3 cells nor NIH3T3 cells transfected with pLLTX aloneproduced any tumor. Only SL6 cells produced spontaneous metastases tothe lung. Replating of SL6, SL6ΔT and control cells into 0.5% agaroseshowed that only SL6 cells formed large colonies. To examine theregulation of reporter gene expression by transactivator, SL6 and SL6ΔTcells were assayed for β-galactosidase activity (Table 1). Whentransactivator was present in SL6 cells, expression of reporter gene wasalmost complete by shut off, compared to background control cells; whentransactivator was removed by cre-lox recombination in SL6ΔT cells, thereporter gene was highly expressed. These results indicate thattransactivator generated antisense RNA can effectively inactivate geneexpression.

TABLE 1 Characterization of SL6 Transactivator 3T3− 3T3+ SL6− SL6+β-Galactosidase Activity (U/μg) 9.26^(a) 10.05 1225.80 19.88 Growth in0.5% Agarose — —  20/10^(5b) _1000/10⁵    Tumorigenicity in Nude Mice0/10 0/10 1/10 10/10 Spontaneous Lung Metastasis^(c) 0/10 0/10 0/10 8/10 ^(a)Means of triplicates. ^(b)The colonies formed by SL6 withouttransactivator were significantly smaller than those formed by SL6 withtransactivator. ^(c)Mice were sacrificed at day 32 with lung metastaseswere confirmed by histology.

A genomic southern blot of SL6 cells using an 1.3 kb neo fragment probeshowed a single chromosomal integration of pLLGSV; both the reportergene and the regulated promoter were faithfully duplicated in accordancewith the retroviral life cycle. Northern blotting of poly(A) RNAisolated from SL6ΔT using a 550 by fragment of 5′ β-geo as a probe,showed a major transcript of 7 Kb in length, and two transcripts of 7.5Kb and 6.5 Kb in smaller amount. Hybridization with the cloned geneconfirmed that the 7 Kb and 6.5 Kb transcripts were fusion transcriptsof the reporter gene and mRNA initiated at a chromosomally-locatedpromoter external to the vector. During cDNA cloning (see below), wealso isolated many alternatively spliced cDNA products, in which thesplice acceptor site of the second copy of the reporter gene in theprovirus had been spliced to several cryptic splice donors of the firstreporter gene, and such aberrant splicing may result in multipletranscripts in Northern blots, as has been observed previously(Friedrich and Soriano, 1991, supra).

cDNA Cloning and Sequence Analysis. A biotin labeledoligodeoxyribonucleotide that corresponds to the 5′ end of β-geo wasused to select β-geo fusion mRNA from SL6ΔT cells by hybridization; thehybridized mRNAs were purified using streptavidin-coated paramagneticparticles, reverse transcribed, converted to double strand cDNA, clonedinto the E. coli plasmid pAmp1, and sequenced by standard methods. Thecloned 120 by cDNA segment contained 70 by of a novel sequence fused inframe to the splice acceptor site 5′ to β-geo. A data base search usingthe BLAST program (Altschul et al., J. Mol. Biol. (1990) 215:403-410)showed 97% identity to a mouse partial cDNA sequence of unknown functionidentified by its expression during differentiation of F9 mouseembryonal carcinoma cells (Nishiguchi et al., (1994) J. Bio. Chem.116:128-139.

A mouse NIH 3T3 cell cDNA library was screened with the 70 by cDNA probeto obtain a full length gene. Four positive clones were isolated, andall contained a 1148 by open translational reading frame (ORF) encodinga predicted 381 amino acid protein of 43,108 kba. The gene defined bythis sequence was designated as tumor susceptibility gene. 101 (tsg101).A potential consensus sequence for initiation of translation, followedby an adenosine residue three bases upstream of a putative ATGtranslation start codon, was located near the 5′ end of the tsg101. Asplice donor consensus sequence (AG) was observed 72 nucleotides intothe cDNA sequence analyzed and four codons downstream of the ATG.

The sequence of full length tsg 101 cDNA and the predicted amino acidsequence of the Tsg101 protein were used to search the non-redundant DNAand protein sequence databases of the National Center for BiotechnologyInformation using the BLAST program. This analysis indicated that aminoacids 231 to 301 of tsg101 are identical, except for two mismatches tocc2, an α-helix domain encoded by a partial cDNA clone identified by itsability to express a protein that interacts with stathmin (Maucuer etal., PNAS USA (1995) 92:3100-3104); an evolutionarily-conservedphosphoprotein implicated in the integration and relay of diversesignals regulating cell growth (Sobel, Trends Biochem. Sci. (1991)16:301-305). The algorithm of Stock and colleagues (Lupas et al.,Science (1991) 252:1162-1164) predicts with a probability of ˜99.8% thatthe helical domain of Tsg101 will form a coiled-coil structure. Aprotein pattern search of full length Tsg101 identified a leucine zipperdomain within the coiled-coil domain of Tsg101, consistent with theobserved ability of the cc2 domain to interact with stathmin..Additionally, seven potential protein kinase C phosphorylation sites(aa11, 38, 85, 88, 215, 225, 357), five potential Casein kinase IIphosphorylation sites (aa38, 210, 249, 265, 290), two potentialN-myristorylation sites (aa55, 156), and three potential N-glycosylationsites (aa44, 150, 297) were present in Tsg101 (Bairoch and Bucher,Nucleic Acids Res. (1994) 22:3583-9). A protein motif search (Prints,Leads University, UK) showed that aa37-46 of Tsg101 resembles thehelix-turn-helix signature domain of the bacteriophage λ repressor(i.e., HTHLAMBDA) (Brennan and Matthews, J. Biol. Chem. (1989)264:1903-1906), and that aa73-83 resembles a fungal Zn-cys bi-nuclearcluster signature (FUNGALZCYS) (Pan and Coleman, PNAS USA (1990)87.2077-2081).

Expression of tsg 101 Sense and Antisense RNA Cause Transformation ofNaive NIH-3T3 Cells. To confirm the role of tsg101 in cell growth, weinvestigated the effects of overexpression of tsg101 in sense andantisense orientations in naive NIH 3T3 cells. in both instances, thetsg101 sequence was expressed in stably transfected cells under controlof the cytomegalovirus (CMV) promoter. Expression of tsg101 in eitherthe sense or antisense orientation resulted in transformation of naiveNIH3T3 cells, as indicated by the ability to form colonies on 0.5%agarose. Whereas no colonies were observed in cells transfected with thevector lacking the insert or in mock transfected cells.

Experimental Procedures

Construction of Vectors. To construct the self-inactivated retroviralgene search vector pLLGSV, a 4.3 kb Xhol-Xhol fragment from pSAβ-geo(Friedrich and Soriano, Genes & Develop. (1991) 5:1513-1523), containingβ-geo reporter gene and a splice acceptor sequence 5′ to the reporter,was ligated into a Xhol linker site of pACYC184 plasmid (Chang andCohen, J. Bacteriol. (1978) 134:1141-1156) that had been digested withTth111l and Xbal. The Nhel site of pACYC was then deleted and the Xholsite 5′ to the β-geo reporter gene was converted into a Nhel site bylinker insertion; a 1.45 kb Pvull-Stul fragment containing 14 lacoperator repeats and a SV40 minimal promoter sequence from pL14CAT(Labow et al., 1990, supra) was introduced into an Spel 5′ to the spliceacceptor site and β-geo in the opposite orientation to β-geo. Thepolyadenylation signal of β-geo was deleted by Xbal digestion andreplaced with a Nhel linker. This 5.4 kb Nhel-Nhel fragment was thenligated in the same orientation as retroviral transcription, into a Nhelsite at the deleted 3′ LTR of pHHAM (Hawley et al., PNAS USA (1987)84:2406-2410) after Nhel partial digestion.

The transactivator vector pLLTX was derived from pHCMVLAP348 (Labow etal., Mol. Cell, Biol. (1990) 10:3343-3356). The Hindlll site at the 3′end of the transactivator was first deleted and a 1952 by Sfil fragmentcontaining a HyTK gene expression cassette (Lupton et al., Mol. Cell.Biol. (1991) 11:3374-3378), was ligated into the Hindlll site upstreamof transactivator to yield pLAPHyTK. A 200 by DNA fragment containingtwo directly repeated loxP sites derived from pBS30 (Sauer andHenderson, Nucleic Acids Res. (1989) 17:147-161) was introduced into aC/al site of pLAPHyTK to give pLLTX. pBS30 was first digested with Sa1land BamHl, and ligated with a Hindlll linker; then the vector wasdigested with Aatll and Xhol to generate this 200 by fragment with twodirectly repeated loxP sites. This 200 by fragment was ligated into aClal site of pLAPHyTK to give pLLTX.

To construct the expression vector pLLEXP I, a 1410 by fragment[containing a human β-actin promoter, the puromycin resistance gene pac,and an SV40 poly(A) site] was first cloned into the BamH1 site of pBR332to generate pBR-β-pac. The Sfil fragment containing the HyTK gene,expression cassette (Lupton et al., 1991, supra) was then inserted intoa BamHl site of pBR-β-pac, after BamHl partial digestion to givepBR-β-pac-HyTK. The expression vector pLLEXP I was generated by Nhel andBglll digestion of pBR-β-pac-HyTK to remove the HyTK gene and replacedby cDNA inserts.

Cell Culture and Transfection. NIH 3T3 cells (ATCC) and GP+E-86 cells(Markowitz et al., J. Virol. (1988) 62:1120-1124) were cultured inDubecco's modified Eagle's medium (DMEM) supplemented with 10% calfserum (3T3) or 10% new born calf serum (GP+E-86), 100 U/m1 penicillin,and 100 mg/ml streptomycin. DNA transfection was carried out byelectroporation (Potter et al., PNAS USA (1984) 81:7161-7165) usingCell-Porator Electroporation systems I (Life Technologies, Inc.) andLipofectamin (Life Technologies, Inc.) according to the protocol of themanufacturer.

Retroviral Infection of Mouse Fibroblast NIH3T3 Cells. To generateinfectious retrovirus, pLLGSV was linearized by treatment with Scal andtransfected into helper cell line GP+E-86 by electroporation. Thetransfected GP+E-86 cells were replated on day 3 and selected with 800μg/m1 G418 for 2-3. weeks. All G418 resistant clones were isolated andexpanded in 24-well plates. Culture supernatant from each clone wasincubated with NIH 3T3 cells in the presence of polybrene (8 μg/m1) for8 hr, and the frequency of integration behind the chromosomal promoterwas subsequently determined by X-gal staining of the infected NIH 3T3cells. The helper cell clones giving the highest frequency ofintegrations behind chromosomal promoters were expanded and culturesupernatant was collected for large scale infection of NIH 3T3 cells.

Isolation of Transformed Clones and Tumorigenicity Assay. Cultures ofG418 resistant NIH 3T3 cells were trypsinized and transfected withHindlll linearized pLLTX DNA by electroporation. The transfected cellswere selected with 500 μg/m1 of hygromycin for 12-18 days. Allhygromycin resistant clones were plated into 0.5% agarose (Li et al., J.Natl. Cancer Inst. (1989) 81:1406-1412), 4 to 6 weeks later, thecolonies formed in 0.5% agarose were isolated and expanded to celllines. To assay the tumorigenicity of the transfected cells, 10⁵ cellswere injected into nude' mice (NIH nu/nu, female and 6 weeks of ag)subcutaneously over the lateral thorax. The animals were examined twiceweekly and sacrificed five weeks later. The neoplastic nature of localtumors and lung metastases were confirmed by histologic examination(Fidler, Cancer Metastasis Rev. (1986) 5:29-49).

cDNA Cloning and Screening of cDNA Library. A biotin labeledoligodeoxyribonucleotide (27 mer) that corresponds to the 5′ end of theβ-geo reporter gene was hybridized with polyadenylated mRNA from SL6ΔTcells, and captured with Streptavidin paramagnetic particles (Promega).The oligo-hybridized mRNA was eluted and reverse transcribed with a genespecific primer corresponding to a sequence located upstream of thebiotin labeled oligo into first strands of cDNA. A uracil DNAglycosylase (UDG) cloning site (Booth et al., Gene (1994) 146:303-308)was incorporated into the gene specific primer to facilitate cDNAcloning. The first strand cDNA was then 3′ tailed with (dG)n by terminaltransferase, and converted into ds cDNA using a UDG-oligo d(c)₂₀ primerand DNA polymerase. The dscDNAs were cloned into the UDG-cloning vectorpAMP1 (Life Technologies, Inc.) and screened for fusion to β-geo. A 70by cDNA segment of novel sequence fused in frame to the splice acceptorsite 5′ to β-geo was used as a probe to screen a mouse NIH 3T3 cDNAlibrary (Stratagene). Positive clones were sequenced with Sequenase 2.0(USB) for both strands.

Southern and Northern Blot Analysis. Genomic DNA was isolated bystandard procedure. Total RNA was isolated with RNA STAT-60 (TEL-TEST),and poly(A) mRNA was isolated with PolyATtract (Promega). Both DNA andRNA blots were probed with PCR generated single-stranded DNA probes.

Example 2

Chromosomal mapping studies assigned TSG101 to human chromosome 11 bandp15, a region showing loss of heterozygosity primarily in breast cancerbut also in other human. malignancies, and proposed previously tocontain tumor suppressor gene(s). Intragenic deletions in TSG101 wereidentified in four of ten metastatic breast cancer cell lines that werestudied. All of these mutations terminated the TSG101 protein-codingsequence before or within the coiled-coil region that interacts withstathmin. These findings support the conclusion that TSG101 is asuppressor of abnormal cell growth and additionally demonstrate thatthis gene has an important role in human breast cancer.

Results

Cloning and Characterization of Human TSG101 cDNA. tsg101 was initiallyidentified in mouse cells by a novel gene discovery approach thatenables regulated functional inactivation of multiple copies ofpreviously unknown genes and selection for cells that show a phenotyperesulting from such inactivation. To obtain TSG101, the human homolog ofmouse tsg101, the 1448 by mouse cDNA sequence was used to query dbEST ofthe National Cancer for Biotechnology Information (NCBI) by the BLASTprogram. Ten human partial cDNA sequences (Expressed Sequences Tags,EST) included in the database showed 85% to 95% identity to mouse tsg101cDNA. A 27 by sequence contained within a region of 100% identitybetween ESTs H53754 and Z30135 was used to design the UDG primers Pa-UDGand Pd-UDG; these primers plus two other UDG primers(Pb-UDG and Pc-UDG)corresponding to sequences bracketing the vector cloning site of aλgt10-based human cDNA library were used to amplify by PCR the 5′(Pc-UDGand Pd-UDG) and 3′(Pa-UDG and Pb-UDG) segments of human TSG101 cDNA,employing total DNA isolated from the human cDNA library as template.The longest 5′ and 3′ PCR products were then joined in the UDG cloningvector pAMPI.

A 1494 by cloned human cDNA insert was termed full length TSG101 cDNA.Sequence analysis of this cDNA identified a 1140 by open reading framepredicted to encode a 380 amino acid protein with a molecular mass of42.841 kDa and a pl of 5.87. The human and mouse cDNAs are 86% identicalat the nucleotide level. The predicted proteins are 94% identical andare distinguished by 20 amino acid mismatches and one gap. A coiled-coildomain (human TSG101 aa 231-302) and a praline-rich domain (human TSG101aa 130-205, 32% proline) typical of the activation domains oftranscription factors are highly conserved between the human and mouseproteins, with only one amino acid mismatch in each of the two domains.The leucine zipper motif in the coiled-coil domain of the human TSG101protein is identical to the one in the mouse protein. Other conservedfeatures identified in human TSG101 include seven putative proteinkinase C phosphorylation sites (aa 11, 38, 86, 89, 215, 225, 357), fivepotential case in kinase II phosphorylation sites (aa 38, 210, 249, 265,290) and three potential N-glycosylation sites (aa 44,150,297). Analysisof the human TSG101 cDNA and protein sequences by the BLAST programsearch of NCBI database did not reveal any significant homology with thesequences for any other human genes.

Expression of TSG101 in human tissues was examined on a multiple-tissueNorthern blot probed with full length tsg101 cDNA. A single 1.5 kbtranscript was observed in all eight human tissues tested and wasslightly more prominent in RNA isolated from liver and pancreas. Thesize of this transcript indicates that the 1494 by cDNA corresponds tofull length native TSG101 mRNA.

Chromosomal localization of human and mouse TSG101 genes. By using PCRprimers that specifically amplify a human TSG101 sequence from the3′-untranslated region, genomic DNA from a panel of 18 human×Chinesehamster hybrid cell lines was analyzed. The expected 210 by PCR productwas obtained only from hybrid cell lines that had retained humanchromosome 11 and from total human genomic DNA, but not from hamsterDNA. The human-specific PCR product was also generated from a cell line(31-2A HAT) that retained only the short arm of chromosome.11 (11p),whereas no PCR amplification was observed using the same primers in acell line that had only the long arm of chromosome 11 (11q). Byconcordant segregation and by excluding all other chromosomes, the humanTSG101 gene is assigned to chromosome arm 11p.

To obtain a human TSG101 genomic DNA probe suitable for mapping byfluorescence in situ hybridization (FISH), the same set of PCR primersemployed for the analysis of hybrid cell lines was used to screen a PAClibrary containing human genomic DNA inserts. Two overlapping clones,PAC1 and PAC2, each containing ˜150 kb inserts, were isolated andconfirmed to contain TSG101 human genomic DNA by Southern blotting usinga 5′ human TSG101 cDNA fragment as probe. Fluorescence in situhybridization of the two PAC clones to human chromosome spreads gaveidentical results, which confirmed the localization of TSG101 onchromosome arm 11p by our somatic cell hybrid analysis. A fluorescencesignal on both chromatids of both copies of chromosome 11 was seen in 20metaphase cells analyzed. Based on the chromosomal R-banding pattern,TSG101 is assigned to chromosome 11 bands p15.1-p15.2.

Radiation hybrid (RH) mapping provides another independent approach tomap human genes and to position them relative to polymorphic markers onthe linkage map. PCR typing for human TSG101 of the Stanford G3 human RHmapping panel revealed a positive result in 11 of the 83 RH cell lines(retention frequency 13.25%). By two point linkage analysis TSG101 wasfound to be closely linked to Sequence Tagged Site (STS) markersD11S921, D11S899, and D11S1308. Both D11S921 and D11S1308 are on theWhitehead Institute integrated map and radiation hybrid map and theirphysical positions approximately correspond to 11p15.

To map tsg101 in the mouse, a mapping panel of 22 mouse×rodent hybridcell lines was analyzed by PCR using mouse gene-specific primers. Thepresence or absence of mouse chromosome 7 in hybrid cell lines was incomplete concordance with the 202 by mouse tsg101 PCR product. All othermouse chromosomes were excluded by at least 3 discordant hybrids. Anattempt to place the gene on the mouse linkage map by typing aninterspecies backcross panel was not successful, as no differencebetween C57BL/6 and M. spretus patterns were detectable by single strandconformational analysis (SSCA) of PCR products. Given the knownconserved syntenic regions on human chromosome 11p and mouse chromosome7, our mapping of the mouse gene provides further evidence that thehuman and mouse sequences we have cloned are true TSG101 gene homologs.

Analysis of TSG101 Mutations in Human Breast Cancers. Extensive studieshave shown deletion or loss of heterozygosity of markers at or near the11 p15 band in a variety of human malignancies, primarily breastcancers, but also Wilms' tumor, and ovarian and testicular malignancies,suggesting that this region contains one or more tumor suppressor genes.Moreover, a region mapping between 11p15.4 and 11pcen was deleted inapproximately 30% of 171 sporadic breast tumors analyzed. The notionthat chromosome 11 contains a tumor suppressor gene specificallyimplicated in the pathogenesis of human breast cancer is supported byevidence that introducing a normal chromosome 11 or segments of thischromosome into breast cancer cells reverses their metastatic potential,as well as other properties associated with oncogenesis. The findingthat homozygous inactivation of tsg101 converts mouse fibroblasts intometastasizing cancer cells suggests that this gene functions as asuppressor of malignant cell growth. To investigate the role for TSG101in human breast cancer, cDNA isolated from ten breast cancer cell lineswas examined specifically for mutations in TSG101, comparing these cDNAswith cDNA obtained from two normal fibroblast strains, two melanoma celllines, and two Wilms' tumor cell lines.

Northern blot analyses showed the presence of a 1.5 kb transcriptcontaining TSG101 in all of the cell lines tested, although the level ofexpression varied among the different lines. By using RT-PCR, theprotein-coding region of TSG101 cDNA corresponding to normal and tumorcell lines was obtained for sequence analysis. In all 16 normal andtumor cell lines, a 1389 by cDNA fragment containing the completeprotein-coding region of TSG101 was amplified. Additionally, in one ofthe breast cancer lines (cell line 4, MDA-MB-231) a smaller cDNAfragment (˜100 by shorter than the 1389 by fragment) was also amplifiedby PCR using the same primers; this fragment (Δ4) was cloned in thepCNTR plasmid vector for sequencing. Sequence analysis revealed a 85 bydeletion, leading to a loss of 28 aa (codons 5-32) and a frameshiftafter codon 32 that causes premature termination of the TSG101 protein10 codons later.

To identify possible deletion mutations in other cell lines, four setsof smaller RT-PCR fragments were studied. Amplification of a 631 byRT-PCR fragment showed a deletion in breast cancer cell line MDA-MB-435(cell line 7), and a 837 by RT-PCR. fragment amplified by-primers P4 andP5 showed a deletion in breast cancer cell line MDA-MB-468 (cell line8). Both deleted RT-PCR fragments (Δ7 and Δ8) were cloned and sequenced.Sequence, analyses showed that Δ7 has a 309 by deletion and Δ8 has adeletion of 457 bp. The deletion in Δ7 (codon 244-347) removes most ofthe coiled-coil domain (aa 231-302) of TSG101; the coiled-coil domain iscompletely deleted in Δ8 (codon 224-376).

To search for mutation(s) in other TSG101 alleles within the cell linescontaining deletions in one allele of TSG101, the cloned 1389 by fulllength RT-PCR fragments from the four breast cancers carrying TSG101deletions (cell lines 4, 6, 7, and 8) were sequenced. The sequencesobtained were compared with the sequences of RT-PCR products fromtranscripts of normal human fibroblasts (cell lines 0 and 1) and humanmelanoma lines (cell line 2 and 3). A point mutation in TSG101 wasidentified in breast cancer cell line 8. This C to T transition resultsin change codon 107 from Trp to Arg. No point mutations in TSG101 werefound in an initial analysis of other tumor cell lines or in the TSG101sequence of melanoma cells or normal fibroblasts.

Genomic Confirmation of Mutation of TSG101 in Breast Cancer Cells. Todetermine the mutations at the genomic level that caused the deletionsobserved in TSG101 cDNA, the corresponding regions of TSG101 genomic DNAwere P CR-amplified using primers derived from intron and exonsequences. A 300 by genomic PCR fragment from cell line 8 and a 1.5 kbfragment frot cell line 7 were sequenced. Sequence analysis confirmedthat the cDNA deletions in the two cell lines results from genomicdeletions.

The extraordinary conservation observed between the mouse and humanTSG101 proteins is consistent with its important biological role. Boththe coiled-coil and proline-rich domains are nearly identical, and thepotential phosphorylation and N-glycosylation sites are completelyconserved between the human and mouse protein. Chromosomal mapping ofTSG101 to human chromosome 11 and mouse chromosome 7, which shareconserved syntenic regions, demonstrate that the human gene and mousegenes are homologs.

Both the mouse and human TSG101 proteins contain a coiled coil domainnearly identical to one previously shown to interact with stathmin, aphosphoprotein proposed to function in the coordination and relay ofdiverse signals regulating cell proliferation and differentiation. Thepresence of multiple DNA-binding domains in the TSG101 protein and aproline-rich domain near the leucine zipper DNA binding motif of thisprotein indicates that the TSG101 gene product is a transcriptionfactor, and therefore a downstream effector of stathmin action.

Two types of TSG101 deletions were observed in breast cancer cells. Onetype involved partial or complete deletion of the coiled coil domain,suggesting a specific functional role of this domain in malignancy. Thesecond type of mutation was a short deletion near the N-terminal end ofthe protein, generating a frame shift from the point of deletion andtermination of the protein by a stop codon 24 aa later. No deletions inTSG101 were found in the normal fibroblast cell lines, melanomas, orWilms' tumors examined.

It is noteworthy that the breast cancer cell lines having a DNA deletionthat contains the TSG101 gene have also been shown to have highmetastatic potential in nude mice. Introduction of a copy of normalchromosome 11 significantly suppressed this metastastic potential. Theseobservations are consistent with the finding that LOH at 11p15 inprimary human breast tumors is associated with poor survival aftermetastasis and the suggestion that LOH at 11p15 is involved in latestage tumor progression.

The TSG101 gene and the protein it encodes are useful for not only thediagnosis of human breast cancer and other human cancers as well, butalso for gaining an increased understanding of mechanisms oftumorigenesis.

Experimental Procedures

cDNA and Genomic DNA Cloning. The two UDG-primers derived from ESTsH53754 and Z30135 were [SEQ ID NO:5] Pa-UDG(5′AGGUCAUGAUUGUGGUAUUUGGAGAUG3′) and [SEQ ID NO:6] Pd-UDG(5′CAUCUCCAAAUACCACAAUCAUGACCU 3′). Two UDG-primers derived from theλgt10 cloning site are [SEQ ID NO:7] Pb-UDG(5′CAUCAUCAUCAUGAGGTGGCTTATGAGTATTTCTTCCAG3′) and [SEQ ID NO:8]Pc-UDG(5′CUACUACUACUACACCTTTTGAGCAAGTTCAGCCTGGTT3′). 5′(Pc-UDG andPd-UDG) and 3′(Pa-UDG and Pb-UDG) segments were amplified by PCR asfollowing condition: 100 μl final volume of 20 mM, Tris-HCl pH 8.55, 3.3mM MgCl₂, 16 mM (NH₄)₂SO₄, 150 μg/ml BSA, 300 μM each dNTP, 1 μl humanplacenta λgt10 cDNA library (titer 10⁶/μl, ATCC), 0.2 μl of KlentagLA(Barnes (1994) P.N.A.S. 91:2216-2220), in a Perkin-Elmer Cetus thermalcycler for 40 cycles of: 95° C. for 45 s (for denaturation), annealingand extending at 72° C. for 1 min. The PCR products were visualized inethidium. bromide-stained low melting agarose gels, purified and clonedinto pAMP1 cloning vector (Life Technologies, Inc.). Multiple cloneswere isolated and both strands of the cDNA inserts were sequenced usingSequenase 2.0 (USB).

The PCR product made using primers, [SEQ ID NO:9] 5′CTGATACCAGCTGGAGGTTGAGCTCTTC3′ -(forward primer) and [SEQ ID NO:10]5′ATTTAGCAGTCCCAACATTCAGCACAAA3′ - (reverse primer) were used to screena PAC library containing human genomic DNA insert (Genome Systems,Inc.), yielding two overlapping clones, PAC1 and PAC2, each containinginserts about 150 kb long. The presence of TSG101-specific sequenceswithin these inserts was confirmed by Southern blotting, using a 5′fragment of human TSG101 cDNA as probe.

Cell Lines and Cell Culture. Human breast cancer cell lines (MDA-MB-231,MDA-MB-436, MDAMB-435, MDA-MB-468, MDA-MB-157, MDA-MB-175VII, MDA-MB361,BT-483, and MCF-7), Wilms tumor cell lines (G401 and SK-NEP-1), andprimary cultures of human normal fibroblast (CCD-19Lua and MRC-9) wereobtained from American Type Culture Collection. Two melanoma cell lines(A375P and A375SM) were provided by I. J. Fidler. All cell lines werecultured in Dulbecco's modified Eagle's medium supplemented with 10%fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin,except for breast cancer BT-483 cells, which were cultured in RPMI-1640medium with 20% fetal bovine serum and two VVilms tumor cell lines (G401and SK-NEP-1), which were cultured in McCoy's 5a medium with 10% fetalbovine serum.

Northern Blot Analysis. A Northern blot filter of multiple normal tissuemRNA was purchased (Clontech). Radioactively-labeled single anti-sensestrand DNA probe generated from full length human TSG101 cDNA by 40cycles of primer extension, using [³²P]dCTP, was hybridized to thefilter using standard methods. The same blot was stripped and hybridizedwith a human β-actin probe synthesized by random priming as an internalloading control.

Somatic cell hybrids, PCR amplifications, and SSCA. The human TSG101gene was localized to a human chromosome using a panel of 18human×Chinese hamster hybrid cell lines derived from several independentfusion experiments (summarized in Francke et al. (1986) Cold SpringHarb. Symp. Quant. Biol. 2:855-866). The mouse tsg101 gene was mapped byanalyzing a mapping panel of 20 mouse×Chinese hamster and two mouse×ratsomatic cell hybrid lines derived from four independent fusionexperiments, as described previously in Li et al. (1993) Genomics18:667-672. The PCR primers used to amplify human and murine TSG101sequences were derived from the 3′ -untranslated region: the humanprimers were those employed to clone TSG101 genomic DNA as describedabove. The murine primers were: [SEQ ID NO:11]5′GAGACCGACCTCTCCGTAAAGCATTCTT3′-(forward primer) and [SEQ ID NO:12]5′TAGCCCAGTCAGTCCCAGCACAGCACAG-(reverse primer). PCR conditions were 95°C., 2 min; then 35 cycles of 94° C., 30 seconds; 68° C., 30 seconds; 72°C., 1 min; followed by 72° C., 7 min. To distinguish the PCR productsfrom human and hamster sequences in some of hybrid lines, single-strandconformation analysis (SSCA) was carried out as described previously inLi et al. (1996) Cell.

Fluorescence in situ hybridization. The chromosomal localization of thehuman TSG101 gene was independently determined by fluorescence in situhybridization (FISH). Two genomic PAC1 and PAC2 clones carrying ˜150 kbinserts, each containing overlapping human TSG101 sequences, werelabeled with biotin-1 1-dUTP by nick-translation using commercialreagents (Boehringer Mannheim). Each labeled probe was hybridized at aconcentration of 200 ng/50 μl per slide to pre-treated and denaturedmetaphase chromosomes from human lymphocytes. Hybridization, signaldetection and amplification, as well as microscopy analysis and digitalimaging were performed as previously described in Li et al. (1995)Cytogenet, Cell Genet. 68: 1 85-191.

Human radiation hybrid mapping panel. The Stanford G3 radiation hybrid(RH) mapping panel was purchased from Research Genetics, Inc. and wasused to further define the localization of the human TSG101 gene onhuman chromosome 11. This panel consists of 83 RH clones of the wholehuman genome with a resolution of approximately 500 kb. All 83 RH celllines were typed for the human TSG101 gene by. using primers and PCRconditions as described above. The results were sent to Stanford HumanGenome Center for analysis with a software package of two-point andmultipoint maximum likelihood methods, described by Boehnke et al. 1991.

RT-PCR and Sequencing of cDNAs. Total RNA was isolated using RNA Stat-60(TEL-TEST). 10 μg of total RNA was treated with 10 units of RNase-freeDNase I (Boehringer Mannheim) for 10 min, extracted withphenol-chloroform twice, and precipitated with ethanol. First strandcDNAs were synthesized by SuperScript RNase II™ reverse transcriptase(Life Technologies) using the TSG101-specific primer [SEQ ID NO:13] P2(5′ATTTAGCAGTCCCAACATTCAGCACAAA3′) and the human GAPDH. antisense primer[SEQ ID NO:14] (5′GTCTTCTGGGTGGCAGTGATGGCAT3′) as a control. 1-2 μl ofeach product was used for PCR amplification with primer sets indicated.Primers used were [SEQ ID NO:15] P1 (5′CGGGTGTCGGAGAGCCAGCTCAAGAAA3′),[SEQ ID NO:16] P3 (5′CCTTACCCACCTGGTGGTCCATATCCTG3′), [SEQ ID NO:17] P4(5′CCTCCAGCTGGTATCAGAGAAGTCGT3′) and [SEQ ID NO:18] P5(5′CACAGTCAGACTTGTTGGGGCTTATTC3′). PCR amplifications were carried outin 50 μl final volume of 20 mM Tris-HCl pH 8.55, 3.3 mM MgCl₂, 16 mM(NH₄)₂SO₄, 150 μg/ml BSA, 300 μM each dNTP, 0.2 μl of KlentagLA (Barnes,supra.), in a Perkin-Elmer/Cetus thermal cycler for 35 cycles of 95° C.for 45 s(for denaturation), 65° C. for 30s (for annealing) and extensionat 72° C. for 30 s to 1 min and 30s. The PCR products were visualized inethidium bromide-stained low melting agarose gels, gel fragments werepurified (Qiagen) and cloned into pCNTR cloning vector (5 Prime-3 Prime,Inc.) Multiple clones were isolated and sequenced using Sequenase 2.0(USB).

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1-23. (canceled)
 24. A complex comprising the polypeptide of SEQ. ID NO: 21 and an antibody bound thereto.
 25. The complex of claim 24, wherein said antibody is a monoclonal antibody.
 26. An antibody which binds to the polypeptide of SEQ ID NO: 21, to form the complex of claim
 24. 27. The antibody of claim 25 wherein said antibody is a monoclonal antibody. 